a) Field of the Invention
The present invention relates to the field of sensitizing human and animal target cells to external dead inducer. The invention particularly relates to method and a composition for inhibiting or neutralizing DNA sequences and corresponding polypeptides thereof, therefore causing sensitization of cells to subsequent treatment by external killing elements, such as chemical entities or radiations.
b) Description of the Prior Art
Post-translational modifications regulate key functions performed by specialized proteins. Poly(ADP-ribosyl)ation, an emerging post-translational modification, is thought to regulate many nuclear functions, including DNA repair, replication and transcription, chromatin structure and apoptosis. Poly(ADP-ribosyl)ation resembles phosphorylation in that: 1) functional groups are added to certain amino acids residues of their target proteins, and 2) both involved two groups of enzymes working in tandem. Poly(ADP-ribosyl)ation metabolism is made possible by the concerted action of poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG).
Different members of the PARP family have been described in the art, such as canonical PARP-1, PARP-2, PARP-3, VPARP, tankyrase-1 and -2, and TiPARP. The PARPs enzymes catalyze the transfer, polymerization and branching of ADP-ribose moiety from NAD+ into poly(ADP-ribose) polymers. The substrate NAD+ is used, in this case, as a building block rather than as an electron transporter. Polymers of broad length ranging from 2 to hundreds of units of ADP-ribose are covalently attached to glutamate residues of many proteins such as histones, PARPs themselves and topoisomerases. PARP-1 and PARP-2, both nuclear enzymes, have their enzymatic activities dramatically stimulated by DNA strand breaks. Although PARP-1 has been shown to be involved in base excision DNA-repair, it has been demonstrated that PARP-1 may not be essential for single-strand breaks repair but might well be involved in the expression of essentials DNA-repair factors. The function(s) of tankyrases is (are) still debated and may either be involved in telomeres metabolism or vesicular transport or both. The roles and function of others PARP members are still under investigation.
Catabolism of poly(ADP-ribose) is conveyed mainly by PARG and, to a lesser extent, by the enzyme ADP-ribosyl protein lyase. The latter catalyzes the removal of the most proximal ADP-ribose residue on the modified protein and seem to be the rate-limiting step of poly(ADP-ribose) degradation. PARG activity was first observed in calf thymus extracts, as suggested by the cleavage of the ribose-ribose bond of pADPr. PARG as known in the art also hydrolyzes the branch points of pADPr. Nonetheless, PARG is specific to pADPr as it cannot hydrolyze the ribose-ribose bond of Ado(P)-Rib(P) or Ado(P)-Rib(P)-Rib(P). The half-life of pADPr is less than one minute in cells treated with alkylating agents such as MNNG. The polymer is thus quickly processed by PARG through endo- and exoglycosidic modes of action. The recent cloning of PARG in human, bovine, mouse, and rat provided the first evidence that the PARG enzyme was synthesized as a single polypeptide. Immunolocalization of PARG enzyme shows it to be at a perinuclear location in the cytoplasm away from the main cellular site of poly(ADP-ribosyl)ation, the nucleus, where PARP-1 and PARP-2 reside. This suggests that catabolism of poly(ADP-ribose) may involve components of the nuclear import/export system.
Little is known on the physiological role of PARG. Studies with the PARG inhibitors gallotanin and nobotanin B showed a protective effect on oxidative and excitotoxic neuronal death. This suggests that PARG is an activator of cell death in certain cell type. A mutation called tej, localized in the Arabidopsis thaliana PARG gene, was shown to interfere with the circadian cycle. This study showed for the first time a relationship between poly(ADP-ribosyl)ation and pace regulation of circadian oscillator.
PARP inhibitors have been reported to be effective in radiosensitizing hypoxic tumor cells and also effective in preventing tumor cells from recovering from potentially lethal damage of DNA after radiation therapy, presumably by their ability to prevent DNA repair.
U.S. Pat. No. 5,032,617, U.S. Pat. No. 5,041,653 and U.S. Pat. No. 5,215,738 disclose compositions and methods for radiosensitizing hypotoxic tumor cells by using benzamide derivatives and nicotinamide compounds. Nevertheless, data are given only on survival rate of tumor cells having been prior sensitized and irradiated in vivo. It is not possible to know whether the survival of animals treated for killing tumor cells was higher than animals not having been treated.
U.S. patent application Ser. No. 200030078212 discloses a pharmaceutical composition containing PARP inhibitors and their uses, resulting in the prevention of a disease resulting from cell damage or death.
According to the state of the art described above, there are still needs to be provide new methods and compositions allowing the targeted killing of cells in an animal or a human.